In vivo identification of an HLA‐G complex as ubiquitinated protein circulating in exosomes

The nonclassical human leukocyte antigen‐ G ( HLA ‐ G ) is a tolerogenic molecule that can be released to the circulation by expressing cells. This molecule can form dimers but some other complexed HLA ‐ G forms have been proposed to be present in vivo. Here, we further characterized these other complexed HLA ‐ G forms in vivo. Ascitic and pleural exudates from patients were selected based on positivity for HLA ‐ G by ELISA . Complexed HLA ‐ G was detected in exosomes, which indicates an intracellular origin of these forms. 2 D ‐ PAGE analysis of exudates and isolated exosomes showed that these high molecular weight complexes were more heterogeneous than the HLA ‐ G 1 expressed by cell cultures. Treatment with deglycosylating enzymes did not change the molecular weight of HLA ‐ G complexes. Immunoblot analysis of exudates and exosomes with an anti‐ubiquitin antibody showed that at least some of these structures correspond to ubiquitinated HLA ‐ G . HLA ‐ G ubiquitination could be reproduced in vitro in HLA ‐ G 1‐transfected cell lines, although with a lower modified/nonmodified protein proportion than in exudates. In summary, we demonstrate new circulating HLA ‐ G forms in vivo that open a new perspective in the study of HLA ‐ G function and analysis.